QUANTIFICATION OF SPECIFIC IMMUNOLOGICAL REACTIONS BY ATOMIC-FORCE MICROSCOPY

The aim of this work is to demonstrate the ability of atomic force mic roscopy (AFM) to detect and to quantify specific immunological reactio ns between antibodies and antigens, with a view to creating a very sen sitive biosensor. A monolayer of antiferritin antibodies was adsorbed onto alkyl silane modified silicon oxide substrates, which were charac terized by X-ray photoelectron spectroscopy (XPS) and contact angle me asurements. The sensitivity limit for antibody detection was quantifie d by radioimmunoassay (RIA) and compared to that obtained by enzyme li nked immune sorbent assay (ELISA) and by AFM after antibody binding wi th colloidal gold labeled conjugates. In this latter case, substrate m odification after reaction was checked by measuring the surface roughn ess (R-rms) variations. AFM was found to be more sensitive than RIA, w ith a detection limit of 0.3 x 10(-3) ng of antibodies per mm(2). Then , the biosensor performance was investigated using ferritin solutions of various concentrations: the antibody/antigen reaction was quantifie d by directly detecting the antigen and measuring surface roughness mo difications. Results were compared to sandwich immunoassay techniques. Up to now, AFM has detected a minimum ferritin concentration of 0.06 mu g/mL.